Low-dose AAV-CRISPR-mediated liver-specific knock-in restored hemostasis in neonatal hemophilia B mice with subtle antibody response

Here, the authors develop an AAV-CRISPR mediated somatic knock-in. They apply this system to restore hemostasis in neonatal hemophilia B mice and show liver-specificity of the knock-in and low serum antibody production. AAV-delivered CRISPR/Cas9 (AAV-CRISPR) has shown promising potentials in preclinical models to efficiently insert therapeutic gene sequences in somatic tissues. However, the AAV input doses required were prohibitively high and posed serious risk of toxicity. Here, we performed AAV-CRISPR mediated homology-independent knock-in at a new target site in mAlb 3'UTR and demonstrated that single dose of AAVs enabled long-term integration and expression of hF9 transgene in both adult and neonatal hemophilia B mice (mF9 -/-), yielding high levels of circulating human Factor IX (hFIX) and stable hemostasis restoration during entire 48-week observation period. Furthermore, we achieved hemostasis correction with a significantly lower AAV dose (2 x 10(9) vg/neonate and 1 x 10(10) vg/adult mouse) through liver-specific gene knock-in using hyperactive hF9(R338L) variant. The plasma antibodies against Cas9 and AAV in the neonatal mice receiving low-dose AAV-CRISPR were negligible, which lent support to the development of AAV-CRISPR mediated somatic knock-in for treating inherited diseases.

Automated summary

The authors developed an AAV-CRISPR mediated somatic knock-in method and applied it to treat hemophilia B. The results showed liver-specificity of the knock-in and low serum antibody production, making it a promising approach for long-term hemostasis restoration.